XTT Cell Viability and Proliferation Assay

The XTT cell proliferation assay determines the cell proliferation rate and, conversely, the reduction in cell viability, when metabolic events lead to apoptosis or necrosis. XTT is a highly sensitive and nonradioactive assay.

A-769662 and dorsomorphin were diluted in cell culture media and assay concentrations were freshly prepared. Briefly, 50 µL UM cell suspensions in culture medium containing 3 × 10³ 92-1 cells, 4 × 10³ MP46 cells, 4 × 10³ OMM2.5 cells, or 5 × 10³ Mel270 cells were plated in 96-well flat-bottom culture plates (Corning, MA, USA) and incubated for 12 h to recover from handling. Varying concentrations of A-769662 and/or dorsomorphin in cell culture media were added into each well in triplicate. Cell-free wells were also prepared in order to determine the background absorbance values. UM cells were treated with varying concentrations of A-769662 or dorsomorphin for 48 h at 37°C, 5% CO₂, and 60% humidity. According to the XTT assay (Biological Industries, Kibbutz Beit-Haemek, Israel) protocol, XTT reagent solution and activation solution were defrosted immediately prior to use in a 37°C bath. 0.1 mL activation solution was added to 5 mL XTT reagent to prepare the reaction solution. Then, 50 µL of the XTT reaction solution was added to each well in order to assess cell viability at the end of the 48 h of incubation period with A-769662 or dorsomorphin. Following 4 h of incubation of the cells with the XTT reaction solution, the absorbance values (of each well) were measured at 465 nm in a microtiter plate reader (SpectraMax Plus, Molecular Devices, CA, USA) at 25°C. Cells incubated in culture medium only (without A-769662 or dorsomorphin) served as the control for cell viability. The half maximal inhibitory concentration (IC₅₀) values of A-769662 and dorsomorphin for each UM cell line were estimated by fitting a model with nonlinear regression.